Identification of New Genes Contributing to the Extreme Radioresistance of Deinococcus radiodurans Using a Tn5-Based Transposon Mutant Library

Here, we have developed an extremely efficient in vivo Tn5-based mutagenesis procedure to construct a Deinococcus radiodurans insertion mutant library subsequently screened for sensitivity to genotoxic agents such as γ and UV radiations or mitomycin C. The genes inactivated in radiosensitive mutants belong to various functional categories, including DNA repair functions, stress responses, signal transduction, membrane transport, several metabolic pathways, and genes of unknown function. Interestingly, preliminary characterization of previously undescribed radiosensitive mutants suggests the contribution of cyclic di-AMP signaling in the recovery of D. radiodurans cells from genotoxic stresses, probably by modulating several pathways involved in the overall cell response. Our analyses also point out a new transcriptional regulator belonging to the GntR family, encoded by DR0265, and a predicted RNase belonging to the newly described Y family, both contributing to the extreme radioresistance of D. radiodurans. Altogether, this work has revealed new cell responses involved either directly or indirectly in repair of various cell damage and confirmed that D. radiodurans extreme radiation resistance is determined by a multiplicity of pathways acting as a complex network.本研究开发了一种高效的基于Tn5转座子的体内诱变流程，用于构建耐辐射球菌（Deinococcus radiodurans）的插入突变体库，随后针对γ射线、紫外线（UV）辐射或丝裂霉素C等遗传毒性剂的敏感性开展筛选。在辐射敏感突变体中失活的基因涵盖多种功能类别，包括DNA修复功能、应激反应、信号转导、膜转运、多条代谢通路以及功能未知的基因。值得注意的是，对此前未被报道的辐射敏感突变体的初步表征显示，环二腺苷酸（cyclic di-AMP）信号通路或可通过调控参与整体细胞应答的多条通路，协助耐辐射球菌细胞从遗传毒性应激中恢复。本研究还鉴定出一种新的属于GntR家族的转录调控因子，由DR0265基因编码，以及一种预测的属于新报道Y家族的核糖核酸酶（RNase），二者均对耐辐射球菌的极端辐射抗性具有贡献。综上，本研究揭示了直接或间接参与各类细胞损伤修复的新细胞应答机制，并证实耐辐射球菌的极端辐射抗性由多条构成复杂调控网络的通路共同决定。